Microfluidic systems including porous polymer electrodes

ABSTRACT

Microfluidic devices that incorporate a porous polymer electrode assemblies, including microfluidic device useful for detection of nucleic acids, as well as methods of using the microfluidic devices.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is (1) based upon and claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. 60/735,467, filed Nov. 10, 2005, and (2) a continuation-in-part application of application Ser. No. 11/479,175, filed Jun. 30, 2006; which, in turn, is based upon and claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. 60/695,910, filed Jun. 30, 2005; U.S. Provisional Patent Application Ser. No. 60/699,950, filed Jul. 15, 2005; and U.S. Provisional Patent Application Ser. No. 60/735,467, filed Nov. 10, 2005. These priority applications are hereby incorporated by reference in their entireties for all purposes.

SUMMARY

A variety of assays can be performed using small scale analytical systems, such as microfluidic systems. The sensitivity, portability, and durability of such systems can be enhanced by incorporating a porous polymer electrode as a system component. Porous polymer electrodes combine the favorable conductive properties of a conductive material, with a porous structure. The resulting porous electrode can be used for qualitative or quantitative analysis, and to capture and/or release charged materials, such as nucleic acids. The pores of the electrode matrix may also be filled with nonconductive material, yielding electrodes having a plurality of discrete conductive surfaces. The use of such porous electrodes in microfluidic systems can confer the advantageous properties of the electrode on the resulting device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of an exemplary porous polymer electrode assembly.

FIG. 2 is a partial cross-sectional view of an alternative exemplary porous polymer electrode assembly.

FIG. 3 is a perspective view of the face of another exemplary electrode assembly.

FIG. 4 is a partial cross-sectional view of yet another exemplary electrode assembly.

FIG. 5 is a cross-sectional view of yet another exemplary electrode assembly.

FIG. 6 is a perspective view of yet another electrode assembly.

FIG. 7 is a schematic depiction of an exemplary microfluidic system.

FIG. 8 is a schematic depiction of an alternative selected microfluidic system.

FIG. 9 is a plot showing the electropolymerization of methoxythiophene on reticulated vitreous carbon, as described in Example 1.

FIG. 10 is a cyclic voltammogram showing the cycling of a porous polymer electrode between a positive and neutral charge, as described in Example 1.

FIG. 11 is a fluorescent microgram of an exemplary electrode assembly held at a positive potential, as described in Example 2. The presence of adsorbed DNA on the electrode surface is demonstrated by fluorescent staining of the DNA by YOYO-1 nucleic acid stain.

FIG. 12 is a fluorescent microgram of an exemplary electrode assembly held at a neutral potential, as described in Example 2. There is little or no DNA present on the electrode surface, and only a small amount within the electrode matrix, as demonstrated by fluorescent staining of the DNA by YOYO-1 nucleic acid stain.

DESCRIPTION OF SELECTED EMBODIMENTS

I. Porous Polymer Electrodes

FIG. 1 depicts an exemplary porous conductive polymer electrode assembly 10, as seen in cross-section. The particular electrode assembly of FIG. 1 is cylindrical, although a variety of geometries are suitable for the disclosed electrode assemblies. The electrode assembly includes a porous monolith 12 that provides a matrix for the resulting electrode. Applied to the surface of the porous monolith is a conductive polymer 14. Selected porous polymer electrodes were described in U.S. patent application Ser. No. 11/479,175 of Lau, et al. for POROUS POLYMER ELECTRODES, filed Jun. 30, 2006, hereby incorporated by reference.

Conductive polymer 14 is typically in electrical contact with a source of electrical potential. In one aspect, a conductive layer 16 that is in contact with conductive polymer 14 provides the electrical contact. Conductive layer 16 of electrode assembly 10 encircles the cylindrical electrode assembly itself. The electrical contact may be direct, where conductive layer 16 physically contacts at least a portion of conductive polymer 14, or indirect, such as where porous monolith 12 is itself suitably electrically conductive. Any suitably robust and conductive material can be used to provide an electrical connection between the conductive polymer 14 and a source of electrical potential. Conductive layer 16 is typically a conductive metal, such as for example, gold, platinum, aluminum, nickel, or chromium. In a particular aspect of the electrode assembly, the conductive layer includes gold metal. In an alternative aspect of the electrode assembly, the conductive layer includes platinum.

The electrode assemblies may be fabricated in any of a variety of geometries. Typically, the electrode assembly is microscopically porous. That is, the assembly incorporates a matrix having pores, cavities, or channels 17. Typically the pores or channels range in size from about 2 μm to about 100 μm across, where at least a portion of the matrix surfaces are conductive and/or capable of being charged. The pores, cavities, or channels present in the porous matrix may be manually formed, or may present as a by product of the formation of porous monolith 12. These pores 17 may have a regular or irregular shape, and may be arranged regularly, such as in an array, or in no particular short- or long-range order. Typically, where the electrode assembly is porous, the microchannels 17, which may trace a tortuous path, permit the flow of a fluid through the matrix, so that the fluid is in at least intermittent contact with areas of conductive polymer. The particular porosity of the electrode assembly is dependent upon, and may be tailored by the particular method of preparation used. In one aspect, the porous character of the electrode assembly occurs by virtue of the conductive polymer being applied to a porous monolith 12 having the desired porosity.

Although the components of the electrode assembly may be selected and fabricated so that they possess sufficient strength and integrity for practical use, the durability of the resulting electrode may be improved by the presence of a substrate layer 18, as shown for the planar electrode assembly of FIG. 2. Although the substrate may participate in conducting electrical potential to the polymer 12, typically the substrate provides mechanical integrity to the electrode assembly, and optionally provides a base or foundation for fabrication of the electrode assembly.

Substrate 18 can be formed from a variety of materials. Typically, the substrate is manufactured from a material that is substantially chemically inert, and which may be readily shaped and/or machined. The substrate can include, for example, metal, glass, silicon, or other natural or synthetic polymers. The substrate can be formed into any of a variety of configurations. More particularly, the substrate can be shaped and sized appropriately so that the resulting electrode assembly can be used in conjunction with any of a variety of types of analytical system. For example, the electrode assembly may be used in conjunction with analytical systems that employ capillary channels, microwells, flow cells, or microchannels, among other architectures.

Where a substrate is present, conductive layer 16 is typically deposited on the surface of the substrate so as to form any electrical circuitry necessary for operating the electrode, including one or more electrical connections to a potential source. Application of the conductive layer 16 can be via, for example, electroless plating, electroplating, vapor deposition, spluttering, or any other suitable method of applying a conductive material.

To facilitate a strong interaction between conductive layer 16 and porous monolith 12 or conductive polymer 14, conductive layer 16 may be physically or chemically modified to enhance the interaction with the polymer. For example, where conductive layer 16 is a metallic layer, the metal surface can for example be chemically activated, or physically roughened, or both. In particular, where the conductive layer 16 is a gold metal layer, chemical activation of the gold surface with a thiol compound can be advantageous in attaching subsequent polymer layers. In one aspect, the gold surface can be modified with α-mercapto-PEG-ω-aldehyde that is subsequently treated with 3-aminopropyl methacrylate, resulting in an active surface moiety that can undergo copolymerization during the application of a polymeric porous monolith 12. A variety of sulfur-containing compounds and their derivatives (e.g. thiols or disulfides) can be used to modify the gold conductive surface.

Electrode assembly 10 can include a conductive surface polymer 14 that has been applied to an underlying porous monolith 12. Electrode assembly 10 can be prepared by preparing a porous monolith on conductive layer 16 in such a fashion that the applied porous monolith incorporates the desired topography, i.e. cavities, pores and/or irregularities having the desired size, shape, porosity and arrangement. The porous monolith can then be modified throughout its porous structure via application of the desired conductive polymer 14. The porous monolith may be prepared from conductive or nonconductive material, provided that an electrical connection is provided between the conductive polymer 14 and the conductive layer 16. Where the porous monolith 12 is substantially nonconductive, the porous monolith can be applied so that portions of the conductive layer 16 are exposed, and therefore placed in electrical communication with the conductive polymer 14, for example as shown at 20 in FIGS. 1 and 2.

In some embodiments, the porous monolith can be an electrically conductive material, for example, reticulated vitreous carbon (i.e., porous glassy carbon). Where porous monolith 12 is itself conductive, the porous monolith can serve as a direct electrical connection between conductive polymer 14 and conductive layer 16, and thereby to a source of applied electrical potential.

A selected porous monolith can be prepared from a three-dimensionally porous film of a poly(acrylic acid), or copolymers of a poly(acrylic acid), which can be polymerized in situ and covalently bound to the surface of conductive layer 16.

The exemplary porous polymer monolith film can be prepared by free radical polymerization of selected monomer subunits. Uni-molecular photoinitiators and/or bi-molecular photoinitiators can be used to initiate the polymerization reaction. It can be desirable to utilize a combination of uni-molecular and bimolecular polymerization initiators, as such systems can enable free radical polymerization of vinyl and ethenyl monomers even in the presence of oxygen.

For example, a suitable porous polymer monolith can be prepared by polymerization of a mixture of acrylic acid and methylenebisacrylamide can be carried out using a combination of a unimolecular and bimolecular initiators. Suitable unimolecular initiators include, but are not limited to, benzoin esters, benzil ketals; alpha-dial koxy acetophenones, alpha-hydroxy-al kylphenones, alpha-amino al kyl-phosphines, and acylphosphine oxides. Suitable bimolecular initiators typically require a coinitiator, such as an amine, to generate free radicals. Bimolecular initiators include, but are not limited to benzophenones, thioxanthones, and titanocenes.

In one aspect of the electrode assembly, the porous polymer monolith is prepared using phase separation/precipitation techniques to create the desired monolith porosity, and therefore the porosity and/or topography of the resulting electrode surface. Porous poly(acrylic acid) monolith can be precipitated by free radical polymerization in the presence of a porogen (an organic solvent), for example dioxane, heptane, ethyl ether, and methyl ethyl ketone. A thin film of a solution including acrylic acid, methylenebisacrylamide, and uni-/bimolecular photoinitiators in methyl ethyl ketone (MEK) can be photopolymerized using a UV-light source. As the polymerization proceeds, the crosslinked polymer which is not soluble in MEK precipitates (leading to phase separation) forming a porous film. Polymerization and subsequent phase separation can be used to form a polymer monolith having the desired degree of porosity. The porosity and pore size of the resulting polymer monolith can be tailored by the selection of the porogen (solvent), the particular monomer(s) used in the reaction, and the polymerization parameters utilized. The mechanical properties of the porous polymer monolith can also be tailored by the addition of an appropriate crosslinking agent and/or selection of desired co-monomer.

Typically, the mechanical integrity of the porous monolith is enhanced when the porous polymer film is bonded to the substrate covalently. For example, where the substrate is glass, the glass surface can be modified using a reactive silane reagent. By reacting the silanol groups on the glass surface with (3-methacryloxypropyl) methyldimethoxysilane, for example, a polymerizable surface methacryloxy-group is formed that can undergo copolymerization with a vinyl monomer, such as acrylic acid, covalently bonding the porous polymer monolith to the glass substrate.

In another aspect, a suitable porous polymer monolith can be prepared by sintering polymeric microparticles. Suitable microparticles may be commercially available, or they can be prepared beforehand. For example, where the microparticles include crosslinked poly(acrylic acid), suitable microparticles can be synthesized via inverse emulsion polymerization of acrylic acid. The polymerization process can be initiated by a thermal initiator, for example, potassium persulfate. Polymerization can further occur in the presence of a suitable polymerization catalyst, for example tetramethylethylenediamine, among others. Polymerization may also be performed in the presence of a desired crosslinking agent, for example N,N-methylenebisacrylamide, among others. The crosslinked poly(acrylic acid) microparticles can be purified, for example by dialysis, and collected by simple filtration.

To prepare the desired porous monolith, a composition that includes the polymeric microparticles can be coated onto the surface of the desired substrate. Typically, the polymer microparticles are prepared with a sufficient degree of crosslinking that the microparticles sinter, or become a coherent solid, at elevated temperatures to give a porous monolith having the desired porosity. To achieve the desired monolith character, the microparticle formulation can contain a thickening agent to control monolith thickness. The thickening agent can be, for example, a silica thixotropic agent, or a water-soluble polymer such as non-crosslinked poly(vinyl alcohol) or non-crosslinked poly(acrylic acid).

Any suitable process can be employed for applying the microparticle composition and sintering the microparticles. For example, the microparticle composition can be applied by spin casting, dip coating, spray coating, roller coating, or other application methods. The resulting coating is typically dried with application of external pressure at elevated temperature. For example, a pneumatic hot press can be used to sinter the microparticles to form the porous monolith. After the sintering process, any water-soluble thickening agent present can typically be removed by rinsing the porous monolith with water.

A primer can be used to improve the adhesion of the sintered monolith onto the desired substrate. For example, where the substrate is glass, the primer can be a silane-derivatized surface agent. The primer can also be a layer of crosslinked or non-crosslinked poly(acrylic acid), polymerized and chemically bonded to the substrate surface as described above.

Typically, where it is advantageous for the porous polymer electrode to exhibit a more open pore structure, for example in applications where a sample solution flows through the electrode assembly, the more open pore structure resulting from the phase separation/precipitation method of monolith preparation can be preferable.

The polymeric porous monolith formulations described above can offer hydrolytic stability, a high degree of control over the surface characteristics of the porous monolith, and cost-effectiveness. However, a variety of other porous monolith compositions may also be used to prepare a monolith having the desired degree of porosity, and that are suitable for application of an appropriately porous electrode assembly.

For example, the porous monolith may be formed from carbon. Specifically, the porous monolith can be formed from carbon cloth, carbon mat, reticulated vitreous carbon, carbon felt, or other carbon materials. A conductive adhesive can be used to bond the carbon porous monolith to the conductive layer. Any appropriate conductive adhesive can be used, including for example a paste comprising a carbon black powder dispersed in a thick solution of polyvinylidene fluoride (PVDF) in N-methylpyrrolidinone. The conductive layer can include, for example, metallic stainless steel or gold. The conductive surface polymer can then be applied to the porous monolith to form the desired electrode assembly.

The application of the conductive polymer 14 can be facilitated by selecting a porous monolith composition having a surface that will interact with the applied coating. For example, the porous monolith can include appropriate functional groups, such as carboxylic acid groups, among others, so that the applied conductive polymer can interact ionically and/or covalently with the porous monolith to enhance binding.

The conductive polymer can be applied to the porous monolith utilizing chemical oxidation. For example, ferric chloride can be used as an oxidant for the precursors pyrrole and bithiophene, and where the porous polymer monolith exhibits surface carboxylic acid groups, treatment of the porous monolith with ferric chloride typically results in association of the Fe(III) ions with the carboxylate groups. When the resulting ferric-loaded porous polymer monolith is exposed to a solution of an appropriate monomer, such as pyrrole or bithiophene, an oxidized and conductive polymer can be deposited on the porous monolith surface. It should be appreciated that any of a variety of analogous chemical oxidants may be used in this manner. For example, where the porous monolith surface is functionalized with ammonium moieties, sodium persulfate can be bound to the surface via the ammonium groups, and subsequently used to oxidize an applied polymer precursor.

Alternatively, the conductive polymer layer can be prepared electrochemically, either in the absence or in the presence of a chemical oxidant. In particular, where the pores present in the porous polymer monolith expose an underlying conductive layer, the conductive polymer can be grown from the surface of the conductive layer itself, creating an advantageous electrical connection between the conductive layer 16 and the conductive polymer 14. Various counter anions (dopants) can be used in this approach, and “doping-dedoping-redoping” techniques as described by Li et al. (Synthetic Metals, 92, 121-126 (1998)) can be employed to to improve conductivity of the resulting conductive polymer. Where the porous monolith is itself conductive, a conductive polymer can be electrochemically oxidized and deposited on the surface of the porous monolith itself.

The conductive polymer layer can be prepared via the chemical and/or electrochemical oxidation of any appropriate monomer or combination of monomers. As used here, an appropriate monomer is one that, upon oxidation, produces a polymer that exhibits sufficient conductivity to be useful as an electrode surface layer. Typically, the resulting polymer can be oxidized and reduced in a controllable and reversible manner, permitting control of the surface charge exhibited by the polymer. Appropriate monomers include, but are not limited to, acetylene, aniline, carbazole, ferrocenylene vinylene, indole, isothianaphthene, phenylene, phenylene vinylene, phenylene sulfide, phthalocyanines, pyrrole, quinoxaline, selenophene, sulfur nitride, thiazoles, thionaphthene, thiophene, and vinylcarbazole, including their derivatives, and combinations and subcombinations thereof.

In a particular example, a non-conductive polyaniline is synthesized according to the protocol reported by Chiang and MacDiarmid (Synthetic Metals, 13, 193-205 (1986)). The non-conductive polyaniline, which is soluble in N-methylpyrrolidinone (NMP), can be applied to the porous monolith. The coated polyaniline can then be oxidized either electrochemically or chemically to create the conductive polymer layer. The ionic interaction between the conductive polyaniline and the negatively charged porous polymer monolith, as well as physical interlocking, anchors the conductive polymer to the porous monolith surface. Where the porous monolith is functionalized with carboxylic acid groups, these can serve as the counter anion of the conductive polymer. The positive charges on the outer surface of the conductive polymer surface can then be used to attract and/or immobilize negatively-charged analytes, and subsequently neutralized electrochemically, to release the captured analytes.

The porous polymer electrodes described herein typically offer a large electrode surface area. This enhanced surface area can offer advantages in selected applications, as will be discussed below. However, the surface area can also result in the electrode exhibiting a significant background double layer capacitance. Where this background signal is undesirable, it can be attenuated by modifying the surface of the porous electrode so that the electrode includes a plurality of discrete conductive domains, where the domain can be partially or fully isolated by a nonconductive matrix. Such a configuration can isolate the conductive domains, thereby reducing the geometric area of the electrode while still allowing for overlap of the diffusion zones of the respective conductive domains. This can reduce the charging current while still allowing for maximal sampling of the solution phase analyte(s). The resulting electrode offers an effectively large surface area for capture and Faradic signals, but with reduced capacitance and therefore reducing background signal. For example, in some aspects, background signal could be reduced by as much as three orders of magnitude.

In some embodiments, an electrode having a plurality of discrete conductive domains may be prepared by first preparing a porous polymer electrode, as described above, and then filling the pores in the porous electrode assembly with a non-porous and non-conductive material. In one aspect, the pores can be filled with a low viscosity two-part epoxy resin, or a latent cure adhesive, among other formulations. The plurality of conductive domains can then be freed mechanically, for example by polishing, sanding, drilling, or other shaping, to reveal conductive polymer islands within the nonconducting matrix. Such conductive islands can have diameters on the order of nanometers to millimeters.

In one aspect of the electrode, as shown in FIG. 3, a surface of the filled electrode matrix is exposed, resulting in a planar electrode assembly 20. The exposed electrode face 22 includes conductive domains 24 separated by nonconductive material, either a nonconductive porous monolith 26, or nonconductive filler material 28. Although FIG. 3 illustrates certain relative dimensions and distributions for elements 24, 26, and 28, these dimensions and distributions are exemplary, and can be varied according to the needs of the user.

Alternatively, the advantages of having isolated conductive domains and a porous electrode matrix may be achieved by drilling or otherwise machining channels in the filled electrode matrix, to yield a porous electrode assembly 30, as shown in FIG. 4. The resulting channels 32 expose isolated domains of conductive polymer 34 in the nonconductive filler material 36 and porous monolith 37. The channels can be randomly distributed, or placed in a regular array. The resulting electrode assembly permits the flow of a sample of interest through or past the electrode, similar to the above-described porous electrode assemblies, with the additional advantage of reduced background signal.

In another example, if the voids 17 of porous polymer electrode 10 of FIG. 1 were filled with a nonconductive filler material, as discussed above, and the upper and lower faces of the electrode were covered as well, a porous electrode matrix could be prepared by machining channels through the cylindrical matrix, as shown in a cross-sectional view in FIG. 5. Electrode matrix 40 includes a nonconductive porous monolith 41, coated with conductive polymer 42, and the resulting voids are filled with nonconductive filler 43. At least a portion of conductive polymer 42 is in electrical contact with a conductive layer 44. Channels 46 extend along the cylindrical axis of the electrode assembly, exposing at least a portion of the conductive polymer 42 on the inner surfaces of the channels, and permitting solution to flow through the electrode assembly. The electrode matrix can include an array of channels having any suitable shape, number of channels, and array geometry.

As an alternative to machining, a nonconductive filler material may include a negative photoresist material. In this aspect, illumination and development of the negative resist in selected areas can also expose isolated conductive islands.

In an alternative aspect, as shown in FIG. 6, an electrode assembly 47 can include an array of conductive porous polymer electrode plugs 48, prepared within apertures or cavities formed in a nonconductive substrate 49. This type of electrode assembly may be prepared by polymerizing a porous electrode matrix as described above, within an appropriate cavity or hole in the nonconductive substrate. Electrode assembly 47 can also incorporate a conductive material in electrical connection with the porous polymer electrode plugs (not shown), for example including copper, gold, or other sufficiently inert and conductive material.

II. Exemplary Applications of the Porous Polymer Electrode

The porous polymer electrode assemblies described herein can possess a variety of advantageous properties in electrochemical applications, including but not limited to applications in potentiometry, voltammetry, polarography, and conductimetry. In particular, the irregular and customizable topography of the electrode surface permits researchers to investigate a variety of bioelectronic phenomena. Additionally, the surface of the porous polymer electrode can be readily customized by either the selection of an appropriate monomer precursor, or by chemical modification of the surface, or both, as is readily understood in the art.

The porous polymer electrodes can facilitate detection, quantitation, immobilization, characterization, and/or purification of an analyte. The porous polymer electrodes can be utilized in vivo or in vitro. Typically, the porous polymer electrodes are useful in a method that includes contacting the electrode with the analyte of interest, and applying an electrical potential to the electrode.

Where the porous polymer electrode is utilized in combination with a selected analyte, the analyte is typically a charged species, or can be oxidized or reduced to generate a charged species. By varying the potential of the porous polymer electrode, the charged analyte species may be captured and/or concentrated and/or released. Typically, the porosity of the electrode matrix is selected to complement and spatially interact with the desired charged analyte. That is, the cavities present on the electrode surface are appropriately sized to accommodate the charged analyte. Preferably, the electrode topography is selected so that the charged analyte interacts with the electrode with some selectivity. The porous polymer electrode can therefore facilitate the capture of the desired analyte, independent of the diffusion direction, and can offer improved detection sensitivities.

Any analyte with an appropriate charge, size and shape can be an appropriate analyte for the disclosed electrodes, including analytes that are modified to include an electrochemically active tag that is either covalently or noncovalently associated with the analyte. Typically the analyte is a biomolecule. The biomolecule may be positively or negatively charged, and can include, for example, polypeptides, carbohydrates, and nucleic acid polymers.

With particular respect to analytes that are nucleic acid polymers, the nucleic acid polymer can be present as nucleic acid fragments, oligonucleotides, or larger nucleic acid polymers including polymers exhibiting secondary or tertiary structure. The nucleic acid fragment can contain single-, double-, triple-, and/or quadruple-stranded structures. The nucleic acid may be a small fragment, or can optionally contain at least 8 bases or base pairs. The analyte can be a nucleic acid polymer that is RNA or DNA, or a mixture or a hybrid thereof. Any DNA is optionally single-, double-, triple-, or quadruple-stranded DNA; any RNA is optionally single stranded (“ss”) or double stranded (“ds”). The nucleic acid polymer can be a natural polymer (biological in origin) or a synthetic polymer (modified or prepared artificially).

Where the nucleic acid polymer includes modified nucleotide bases, the bases can include, without limitation, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2′-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2′-O-methyl pseudouridine, beta-D-galactosylqueuosine, 2′-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyl adenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methyl cytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, beta-D-mannosylqueuosine, 5-methoxycarbonylmethyl -2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine 2-methylthio-N6-isopentenyladenosine, N-((9-beta-D-ribofuranosyl-2-methylthiopurine -6-yl)carbamoyl)threonine, N-((9-beta-D-ribofuranosylpurine-6-yl)N-methylcarbamoyl) threonine, uridine-5-oxyacetic acid-methylester, uridine-5-oxyacetic acid, wybutoxosine, pseudouridine, queuosine, 5-methyl-2-thiouridine, 2-thiocytidine, 5-methyl -2-thiouridine, 2-thiouridine 4-thiouridine, 5-methyluridine N-((9-beta-D-ribofuranosylpurine -6-yl)-carbamoyl)threonine, 2′-O-methyl-5-methyluridine, 2′-O-methyluridine, wybutosine, 3-(3-amino-3-carboxy-propyl)uridine, and (acp3)u.

The nucleic acid polymer analyte is optionally present in a condensed phase, such as a chromosome. The nucleic acid polymer optionally contains one or more modified bases or links or contains labels that are non-covalently or covalently attached. For example, the modified base can be a naturally occurring modified base or a synthetically altered base. The nucleic acid polymer can also be, or can include, peptide nucleic acids such as N-(2-aminoethyl)glycine units. The nucleic acid polymer can be modified by a reactive functional group, or be substituted by a conjugated substance. In one aspect, the nucleic acid polymer is modified by the association of an electrochemically active tag for electrochemical detection.

The analyte solution can be, or can be derived from, a biological sample that is prepared from a blood sample, a urine sample, a swipe, or a smear, among others. Alternatively, the sample may be an environmental sample that is prepared from an air sample, a water sample, or a soil sample, among others. The analyte solution can be obtained by extraction from a biological structure (e.g. from lysed cells, tissues, organisms or organelles). The sample typically is aqueous but can contain biologically compatible organic solvents, buffering agents, inorganic salts, and/or other components known in the art for assay solutions.

The analyte of interest is typically present in an aqueous, mostly aqueous, or aqueous-miscible solution prepared according to methods generally known in the art. Any method of bringing the analyte solution into contact with the porous polymer electrode is generally an acceptable method of bringing the analyte into contact with the electrode. In one aspect, the electrode is immersed in the analyte solution. In another aspect, the analyte solution is applied to the electrode. Where the electrode is incorporated in an apparatus or device, the apparatus or device can include suitable fluidics for preparing the analyte solution and/or bringing the analyte solution into contact with the electrode. A chromatographic column can be placed upstream from the porous polymer electrode, and the chromatographic column can be configured to perform one or more of filtration, separation, isolation, and pre-capture/release of biomolecules or cells.

The step of detecting the analyte comprises any method of electrochemically detecting the presence of the analyte at the electrode. Typically, a potential is applied to the electrode surface, or the applied potential is varied, and a resulting current is determined. Alternatively, the potential can be held at a selected value, and a change in current is determined over time, or a constant current can be applied and the resultant voltage determined. The presence of the analyte may be qualitatively detected, or the amount of analyte can be quantitatively determined, typically by comparison with a standard, such as a known amount of the same or similar analyte. Detection and quantitation can be enhanced by the presence of an electrochemical label that is either covalently or noncovalently associated with the analyte.

The correlation generally can be performed by comparing the presence and/or magnitude of the electrochemical response to another response (e.g., derived from a similar measurement of the same sample at a different time and/or another sample at any time) and/or a calibration standard (e.g., derived from a calibration curve, a calculation of an expected response, and/or an electrochemically active reference material).

The high surface area of the disclosed porous polymer electrode may improve analyte detection sensitivity. Particularly where the analyte is a charged analyte, and an appropriate potential is applied to the electrode to capture the analyte. In one aspect, the porous polymer electrode can be used to capture and/or concentrate a charged analyte by electrostatically attracting the analyte to the electrode surface. By capturing the analyte from a flowing sample, for example, the sample can be depleted of analyte. By removing the applied potential, or by reversing the polarity of the applied potential, the captured analyte may be released into solution for collection or further characterization. This is a particularly advantageous application where the analyte is a nucleic acid or nucleic acid fragment.

For example, the charged analyte may be a nucleic acid polymer exhibiting an overall negative charge. By applying a positive charge to the porous polymer electrode, and by selecting an electrode having pores and surface features complementary to the nucleic acid polymer of interest, the nucleic acid polymers can be captured and concentrated at the electrode surface. In one aspect, the porous polymer electrode can be switched between a positively oxidized state and a neutral reduced state, and this reversibility is used to capture and release negatively charged nucleic acid fragments. In some embodiments, the porous polymer electrode can be used to detect and/or quantify nucleic acid fragments resulting from PCR amplification.

A variety of assays for detecting nucleic acid amplification are described in U.S. patent application Ser. No. 11/448,439 to Aivazachvili et al., titled DETECTION OF NUCLEIC ACID AMPLIFICATION, filed Jun. 30, 2006, and hereby incorporated by reference. Selected assays disclosed in the provisional application may be advantageously carried out using a porous polymer electrode as disclosed herein, and in particular may be advantageously carried out using a microfluidic device incorporating a porous polymer electrode, as described below.

Background noise in electrochemical systems comes from inherent background currents in the measurement systems and capacitive charging currents. As these currents can be small, a better signal-to-noise ratio and sensitivity can be achieved with an electrochemical device than in devices utilizing other detection methods. Further, as electrochemical methods typically use small currents and voltages, devices incorporating the porous polymer electrode typically do not require large, expensive, and heavy power supplies. This is an advantage over devices that require light sources for optical detection methods, as an electrochemical-based device typically does not require optical components such as light sources, mirrors, filters, detectors, support mechanics, or movement mechanics. Electrochemical-based devices therefore lend themselves to use in portable and/or handheld devices.

III. Apparatus Incorporating a Porous Polymer Electrode

The polymer electrodes as described above may be incorporated into a variety of apparatus, systems, or devices. The electrode may be incorporated for example as a portion of a microplate, a PCR plate, or a silicon chip. In one aspect, the polymer electrode is incorporated into a device, such that the analyte solution flows past or through the matrix of the porous polymer electrode. In one example, the analyte solution flows through a three-dimensional porous matrix, as for example the cylindrical electrode assembly shown in FIG. 1. Alternatively, the porous polymer electrode is adapted either for immersion in an analyte solution (i.e., a ‘dip stick’), or for the analyte solution to flow past the porous polymer electrode, as for example the planar electrode assembly shown in FIG. 2. The porous polymer electrodes described herein are particularly well suited for incorporation into microfluidic devices.

A microfluidic device is a device that utilizes small volumes of fluid. In some cases, a microfluidic device can utilize volumes of fluid on the order of nanoliters, or less. In one example, a microfluidic device can utilize volumes of fluid on the order of picoliters. Microfluidic devices can utilize a variety of microchannels, wells, and/or valves located in various geometries to prepare, transport, and/or analyze samples. These microchannels, wells and/or valves can have dimensions ranging from millimeters (mm) to micrometers (μm), or even nanometers (nm). Microfluidic devices may also be referred to as ‘mesoscale’ devices, or ‘micromachined’ devices, without limitation. Microfluidic devices can rely upon a variety of forces to transport fluids through the device, including injection, pumping, applied suction, capillary action, osmotic action, and thermal expansion and contraction, among others. In one example, microfluidic devices can rely upon active electro-osmosis to assist in the transport of aqueous samples, reagents, and buffers.

An exemplary microfluidic device 50 is depicted schematically in FIG. 7, and includes a porous polymer electrode assembly 52, a controller 54 configured to control the electrical potential applied at electrode assembly 52, one or more components 56 suitable for preparing a sample solution of interest 58, and fluidic systems 60 suitable for transporting the sample solution of interest 58 to and from the electrode assembly 52.

The flow passages of such a microfluidic device can exhibit cross-sectional dimensions on the order of approximately 0.1 μm to 500 μm, although typical widths are on the order of 2.0 to 300 μm, more preferably 10 to 100 μm. For many applications, channel widths of 5-50 μm can be used. Reaction or mixing chambers fabricated on or in the substrate can have larger dimensions, for example, up to a few millimeters. Generally, the depths of the microfluidic channels and chambers are on the order of 0.1 to 100 μm, typically 2-50 μm.

Typically, a microfluidic device includes a substrate that is microfabricated to define the various channels, mixing and/or reaction chambers and inlets desired for the analysis of interest. The channels, chambers and other features of the device can be designed and fabricated from a solid or semi-solid substrate. Typically, the substrate is silicon, and the microfluidic channels and chambers are microfabricated using established micromachining methods.

The channels and elements of the microfluidic device may be fabricated on the surface of the substrate, and then a cover may be adhered over the substrate surface. Although any suitable cover may be used to seal the substrate surface and define the microfluidic channels and chambers, a transparent cover permits the operation of the microfluidic device to be monitored. Typically, a glass cover is adhered to the substrate. The microfluidic devices described herein are typically configured to analyze sample volumes of less than or about 10 μL.

The transport of fluids throughout the microfluidic device can be determined via visual observation, or by optical detection and analysis, particularly where a transparent cover or transparent substrate are used.

A variety of microfluidic devices are described in U.S. Pat. No. 5,296,375 to Kricka et al. (1994); and U.S. Pat. No. 5,498,392 to Wilding et al. (1996); both hereby incorporated by reference.

The sample of interest may be purified to a greater or lesser extent before being added to the microfluidic device. Alternatively, the microfluidic device can incorporate one or more components configured to prepare the sample for exposure to or analysis by the porous polymer electrode assembly.

Sample preparation steps can include, for example, cell lysis, protein denaturation, polymerase chain reaction (PCR), electrophoresis, affinity chromatography, and electrochemical analysis. Where the sample of interest includes biological materials, pre-treatment of the sample can include one or more procedures such as liquifaction, digestion, and dilution, among others.

Where the microfluidic device is intended to purify an analyte, for example by capturing the analyte and subsequently releasing it, the analyte typically is either charged, or is capable of acquiring a charge, so that it can electrostatically interact with the surfaces of the porous polymer electrode. Where the analyte is not itself charged, the analyte can be combined with a capture probe for the analyte that will complex with the analyte to provide a charged species.

Where the microfluidic device is intended to detect or quantitate the analyte, the analyte is optionally combined with a capture probe capable of specific interaction with the analyte, and that includes a detection reagent. Where detection is accomplished by the porous polymer electrode, the detection reagent is generally an electrochemically active species.

Analysis of the analyte can be combined with additional instrumental analyses, including optical characterization of the analyte. Where the microfluidic device also performs an optical analysis, the analyte can either be detected directly, or can be combined with a capture probe that confers a detectable optical property upon the analyte. For example, a colorimetric or luminescent label may be combined with the analyte, in addition to an electrochemically active label.

Microfluidic devices incorporating a porous polymer electrode can be used to perform any of a variety of assays that take advantage of the advantageous properties of the porous polymer electrode, as described above. In some embodiments, the subject microfluidic device is useful for the detection, quantification, immobilization, characterization, and/or purification of an analyte, particularly where that analyte is a biomolecule, and most particularly where the biomolecules is a nucleic acid polymer.

A representative microfluidic device, suitable for the amplification and subsequent detection of a nucleic acid polymer is shown in FIG. 8. The microfluidic device 62 is depicted schematically, and for the sake of simplicity, is not intended to depict all of the microchannels and wells that may be present in such a microfluidic system. Selected microfluidic devices, including microfluidic devices suitable for amplification and detection of nucleic acid polymers, are described in International Publication No. WO 93/22053 by Wilding et al. (1993); U.S. Pat. No. 5,304,487 to Wilding et al. (1994); and U.S. Pat. No. 5,296,375 to Kricka (1994); each hereby incorporated by reference.

The microfluidic device 62 includes a porous polymer electrode assembly 64, and a controller 66 configured to control the electrical potential applied at electrode assembly 64. The controller typically serves as both a power supply and instrument for performing amperometric or potentiometric measurements.

Upstream from the porous polymer electrode assembly 64 is a sample preparation region 68 of the microfluidic device that is configured to prepare a sample solution of interest. Sample preparation region 68 includes reagent reservoirs 70 configured to supply reagents useful for the sample preparation process. The various chambers of the microfluidic device are interconnected via a microfluidic channel system 72 suitable for transporting reagents, sample solutions, and reaction products through the device, and particularly transport such species to and from the electrode assembly 64.

A sample, typically a biological sample, can be introduced into the microfluidic device via an inlet 74. The sample can be introduced by injection, by electro-osmosis, by capillary action, or any other suitable introduction method. The microfluidic device optionally includes a pretreatment well or chamber 76. Pretreatment chamber 76 permits the biological sample to be mixed with reagents for sample digestion, liquidation, or diluting, if desired. Such pretreatment can be used to render the biological sample fluid enough to enhance the effectiveness of downstream processes.

After this pretreatment, the sample is optionally filtered. For example, the sample can be transported, typically by electro-osmotic pumping, through a filter 78 into a reaction chamber 80. Filter 78 can be used to remove large particles that may interfere with downstream reactions. The filter can be any appropriate filtering agent that is compatible with the biological sample under investigation. For example, filter 78 can include a membrane filter, or a fritted glass filter having a relatively large pore size, for example approximately 100 μm.

Reaction chamber 80 can be used for lysis and denaturing of the sample. As shown in FIG. 7, reagents useful for the lysis and/or denaturing process can be added from reagent reservoir 82 via valve 84. The lysis and/or denaturing process can be accelerated by heating via heating unit 86. Heating unit 86 can include one or more warming lamps, heating coils, fluid heat exchangers, or any other suitable heating apparatus, as well as fans, blowers, heat exchangers, or other suitable cooling mechanism for cooling reaction chamber 80.

After lysis and/or denaturing, the sample is transported to PCR chamber 88, optionally passing through an additional filter 90 en route. Filter 90, when present, is typically finer than filter 78, when present. For example, unlike a relatively coarse filter 78 having a pore size of about 100 μm, filter 90 can be selected for a pore size of approximately 5-10 μm. Such a fine filter can be used to remove undesired byproducts of the lysis/denaturing process. Once the sample has reached PCR chamber 88, reagents useful for the PCR process can be added to PCR chamber 88 from PCR reagent reservoir 92 via valve 94. PCR chamber 88 can be heated by heating unit 96. Similar to heating unit 86, heating unit 96 can be any appropriate heating mechanism for facilitating the PCR process, and typically includes a cooling mechanism, so that heat cycling can be accomplished in PCR chamber 88. Selected suitable thermal cycling mechanisms are described in U.S. Pat. No. 5,455,175 to Wittwer et al. (1995) hereby incorporated by reference.

After PCR is complete, the sample can be transported to electrolysis chamber 97 through another filter 98 having a pore size of approximately 5-10 μm. Electrolysis chamber 97 includes an electrode 100, controlled by a controller. Although depicted as being electrically connected to controller 66 in FIG. 7, the controller for electrode 100 can be the same or different from the controller for porous polymer electrode 64. Appropriate reagents can be added to electrolysis chamber 97 from reagent reservoir 102 via valve 104. The reagents added to the electrolysis chamber can include, for example, a capture probe for the amplified nucleic acid polymer that incorporates a detection reagent.

When present, the capture probe is typically a selective binding partner for the amplified nucleic acid polymer. The capture probe can be from any suitable source and can have any suitable structure. The capture probe can be obtained from a natural and/or artificial source. Accordingly, the capture probe can be synthesized or formed by a cell(s), a cell lysate(s), a synthetic enzyme(s), chemical synthesis, enzymatic cleavage, chemical cleavage, and/or ligation, among others. The capture probe thus can be RNA, DNA, or any suitable analog thereof. Furthermore, the capture probe can belong to the same structural class of molecules as the analyte (e.g., each being DNA or each being RNA) or to a different class of molecules (e.g., the capture probe being a nucleic acid analog and the analyte being RNA or DNA, among others).

The capture probe can have any suitable backbone structure relative to the amplified nucleic acid polymer. In some examples, the capture probe can have a different backbone than the analyte, such as a less charged backbone in the capture probe and a more charged backbone in the analyte (or vice versa). With this arrangement, the amplified nucleic acid polymer can have a greater affinity than the capture probe for the porous polymer electrode, or (vice versa). The analog backbone of the capture probe can lack phosphate moieties, ribose moieties, or both. In some examples, the analog backbone of the capture probe can include a plurality of amide moieties. In some examples, the analog backbone can be a peptide backbone, such that the analog is a peptide nucleic acid. A peptide backbone, as used herein, is any backbone that can be hydrolyzed to release a plurality of amino carboxylic acids, particularly alpha-amino carboxylic acids. In exemplary embodiments, the peptide nucleic acid has a backbone formed of linked N-(2-aminoethyl)-glycine subunits, which position an array of nucleotide bases through methylene carbonyl moieties of the backbone.

The capture probe can be configured to form a duplex with the amplified nucleic acid through base-pair interactions, so that the capture probe and analyte together form an at least partially double-stranded nucleic acid. Accordingly, a section (or all) of the analyte can be complementary to a section (or all) of the analyte. Alternatively, or in addition, the capture probe can include a double-stranded region, independent of the analyte, for example, to couple the capture probe to the matrix of the optical element. The capture probe can be configured to hybridize (base-pair) to any region of the analyte, for example, the capture probe can hybridize adjacent an end or spaced from the end of the analyte.

In one example, a suitable capture probe includes one or more detectable electrochemical labels that can be associated with the capture probe either covalently or noncovalently. The capture probe can further include, without limitation, a luminescent label (including fluorescent, luminescent, and chemiluminescent labels), or a colorimetric label, or a combination thereof. Alternatively, the selected label can be detected indirectly, for example by the interaction of the label with an additional detection reagent.

Where the label interacts with an additional detection reagent, the label is typically a member of a specific binding pair, such as a hapten for a labeled antibody, or a nucleic acid sequence that is labeled by a complementary sequence. The label may include a digoxigenin moiety, for example, that can be used as a target for horseradish peroxidase or alkaline phosphatase detection, followed in turn by chemiluminescent or colorimetric detection. The additional detection reagent can include an electrochemical mediator, so that association of the label with the additional detection reagent facilitates electrochemical detection of the capture probe.

In a particular example, detection of amplification is via electrochemical detection at the porous polymer electrode 64, optionally via the presence of an electrochemical mediator.

In a particular example, detection of amplification utilizes a capture probe that includes a primer modified with an electrochemical label, and a specific complexing protein. Such a capture probe can be added to electrolysis chamber 97, so that the amplified nucleic acid polymer of interest associates with both the labeled primer and the complexing protein to form a complex. After the noncovalent complex is formed, a potential is imposed between electrode 100 in electrolysis well 97 and electrode 106 in electrolysis well 108. Typically, electrode 100 is held at a cathodic potential, and electrode 106 is held at an anodic potential so that, in conjunction with a thin layer of crosslinked polyacrylamide gel 110, electrophoresis occurs across gel 110. While electrophoresis is occurring, the porous polymer electrode is typically either electrically neutral or held in a non-conductive state.

The polyacrylamide gel is typically prepared with a low degree of crosslinking. Under these electrophoretic conditions, all nucleic acid fragments with the exception of target DNA that has complexed and hybridized to the protein and labeled primer, will migrate to electrolysis chamber 108. The relatively large nucleic acid-protein complex is left behind due to its large size and relative inability to penetrate the thin layer of crosslinked polyacrylamide gel.

Although electrophoretic separation has been described, any suitable separation process could be used to isolate the target nucleic acid polymer, including for example, mechanical separation, size exclusion chromatography, separation using derivatized beads or matrix. The matrix can include, for example, magnetic beads or a streptavidin-modified matrix, and can also be used to separate the analyte nucleic acid sequence from other nucleic acid fragments and unbound label.

Once any excess and unbound electrochemically active capture probe and non-target DNA fragments have been removed from the vicinity of the porous polymer electrode assembly 64, an anodic potential can be applied to the electrode assembly 64, while electrode 100 in electrolysis chamber 97 is held at a cathodic potential. If not already conductive, the porous polymer electrode is converted to its conductive state and positively charged.

As the hybridized nucleic acid complex migrates from the thin layer of crosslinked polyacrylamide gel 108, the complex can be electrostatically captured at the positively charged porous polymer electrode, and concentrated on the internal electrode surface. The target nucleic acid polymer can be detected electrochemically if the electrochemical label selected for use is compatible with the material of the polymer electrode. The electrochemical detection of the target nucleic acid sequence depends on the redox potential of the electrochemically active label when associated with the primer, the complexing protein used, and the target nucleic acid polymer.

The specific complexing protein used to form the nucleic acid complex can be selected from any of a group of recombinases, single strand binding proteins, antibodies, transcription factors or any other nucleic acid-binding protein. The binding may also be mediated by one or more additional reagents, including digoxigenin or biotin, among others.

EXAMPLES

The following examples describe selected aspects and embodiments of microfluidic systems including porous polymer electrodes. These examples are included for illustration and are not intended to limit or define the entire scope of the present teachings. In particular, the specific aspects and embodiments disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible.

Example 1

A portion of reticulated vitreous carbon (RVC) foam (average pore size about 60 μm, 12-15% density, DUOCEL), roughly 3 mm×5 mm×15 mm in size, is cleaned by rinsing in acetone and dried under nitrogen. Electrical contact to the foam is achieved using an alligator clip. The RVC electrode is dipped into a stirred solution of 1:3 acetonitrile: deionized water containing 35 mM 3-methoxythiophene and 10 mM sodium perchlorate. The area of the RVC exposed to the solution is roughly 3 mm×5 mm×8 mm. Electropolymerization of the methoxythiophene proceeded at 1.4 V vs. Ag/AgCI for 300 sec using a platinum foil counter electrode. This activation process is shown in FIG. 9. After polymerization, the electrode was removed from the solution, rinsed with water and placed back into a solution of 10 mM sodium perchlorate. Cyclic voltammetry (20 mV/s) was then run to switch the charge state of the conductive polymer coating between positive and neutral as shown in FIG. 10.

Example 2

A porous polymer electrode assembly is prepared by electrochemically depositing positively charged poly(3-methoxythiophene) in its oxidized state on the surface of a monolith of reticulated vitreous carbon, as described in Example 1. The ability of the resulting electrode assembly to capture nucleic acids is verified by exposing the electrode assembly to human genomic DNA that is prestained with the fluorescent nucleic acid stain YOYO-1 (Molecular Probes, Inc., Eugene, Oreg.). As shown in FIG. 11, fluorescent micrograms of the positively charged electrode assembly indicate the presence of human DNA on the surface of the electrode assembly. As a control, the experiment is repeated with the poly(3-methoxythiophene) polymer electrochemically reduced to its neutral state. As shown in FIG. 12, the neutral electrode assembly shows little or no YOYO-1 fluorescence on the surface of the electrode assembly, and only a small amount of fluorescence within the electrode matrix.

The disclosure set forth above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in applications claiming priority from this or a related application. Such claims, whether directed to a different invention or to the same invention, and whether broader, narrower, equal, or different in scope to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure. 

1. A microfluidic device, comprising: a porous electrode assembly that includes a porous monolith; a conductive polymer applied to at least a portion of the porous monolith, so that the surface of the material defines a porous topography; and an electrical connection between the conductive polymer and a potential source; and a controller configured to control the electrical potential applied to the conductive polymer.
 2. The microfluidic device of claim 1, wherein the porous monolith includes a conductive material; and the electrical connection between the conductive polymer and the potential source includes the porous monolith.
 3. The microfluidic device of claim 1, further comprising a fluidic system configured to transport a sample of interest to and from the porous polymer electrode.
 4. The microfluidic device of claim 3, where the fluidic system further comprises one or more filters configured to filter the sample of interest.
 5. The microfluidic device of claim 3, further comprising one or more components suitable for preparing a sample solution for analysis by the porous polymer electrode.
 6. The microfluidic device of claim 5, where the one or more components suitable for preparing a sample solution of interest include one or more reagent reservoirs.
 7. The microfluidic device of claim 5, where the one or more components suitable for preparing a sample solution of interest include a chamber configured for a lysis reaction.
 8. The microfluidic device of claim 5, where the one or more components suitable for preparing a sample solution of interest include a chamber configured for a denaturation reaction.
 9. The microfluidic device of claim 5, where the one or more components suitable for preparing a sample solution of interest include a chamber configured for a polymerase chain reaction.
 10. The microfluidic device of claim 1, wherein the porous monolith includes pores that are about 2 μm to about 100 μm across.
 11. The microfluidic device of claim 1, where the porous polymer electrode assembly comprises: a substrate; a conductive layer disposed on the substrate; a porous monolith disposed on the conductive layer; and a conductive polymer applied to the porous monolith, so that the conductive polymer at least partially defines the pores present in the porous monolith, and so that an electrical connection is formed between the conductive layer and the conductive polymer.
 12. The microfluidic device of claim 1, where the porous polymer electrode assembly comprises: a porous nonconductive monolith; a nonporous and nonconductive filler material within the pores of the porous monolith; and a conductive polymer disposed in the pores of the monolith, and interposed between the monolith and the filler material.
 13. The microfluidic device of claim 12, wherein a surface of the porous electrode assembly exposes a plurality of conductive domains at least partially isolated by nonconductive filler material and nonconductive monolith.
 14. The microfluidic device of claim 13, wherein the conductive domains are exposed on an external surface of the porous electrode assembly.
 15. The microfluidic device of claim 13, wherein the conductive domains are exposed on an internal surface of a channel in the porous electrode assembly.
 16. A microfluidic device for analyzing nucleic acids, comprising a substrate having a plurality of microfluidic chambers and channels fabricated therein; a cover adhering to the substrate surface; an inlet configured to receive a biological sample; one or more chambers configured for pretreatment of the biological sample; one or more chambers configured for subjecting the biological sample to a polymerase chain reaction; one or more chambers configured to separate a nucleic acid polymer amplified by the polymerase chain reaction; and a porous polymer electrode configured to detect the amplified nucleic acid polymer.
 17. The microfluidic device of claim 16, further comprising a chamber configured to associate the amplified nucleic acid polymer with an electrochemically detectable label.
 18. The microfluidic device of claim 16, where the chambers configured to separate the amplified nucleic acid polymer are configured to electrophoretically separate the amplified nucleic acid polymer.
 19. A method of analyzing a sample, comprising: introducing a sample into the microfluidic device of claim 1; preparing the sample for analysis by the porous polymer electrode; transporting the sample to the porous polymer electrode; and applying an electrical potential to the porous polymer electrode.
 20. The method of claim 19, further comprising detecting an analyte of interest in the sample.
 21. The method of claim 20, wherein detecting the analyte of interest includes detecting a charged biomolecule.
 22. The method of claim 20, wherein detecting the analyte of interest includes detecting an electrochemically active tag associated with the analyte of interest.
 23. The method of claim 20, wherein detecting the analyte of interest includes detecting a negatively charged biomolecule, and applying an electrical potential includes applying a positive electrical potential to the porous polymer electrode.
 24. The method of claim 19, further comprising retaining an analyte of interest in the porous polymer electrode due to the applied electrical potential.
 25. The method of claim 24, further comprising applying a second electrical potential to the porous polymer electrode, where the second applied potential releases the analyte of interest from the porous polymer electrode.
 26. A method of detecting a nucleic acid, comprising: introducing a sample thought to contain a target nucleic acid into the microfluidic device of claim 16; pretreating the sample; subjecting the pretreated sample to the polymerase chain reaction; separating amplified nucleic acid polymers from the polymerase chain reaction mixture; and detecting the amplified nucleic acid polymers using the porous polymer electrode.
 27. The method of claim 26, where pretreating the sample includes one or more of digesting, liquidating, diluting, lysing, and denaturing the sample.
 28. The method of claim 26, further comprising labeling the amplified nucleic acid polymers with an electrochemically active label.
 29. The method of claim 28, further comprising labeling the amplified nucleic acid polymers with a specific complexing protein.
 30. The method of claim 26, where separating the amplified nucleic acid polymers includes electrophoretically separating the amplified nucleic acid polymers from the polymerase chain reaction mixture. 